Intestinal apical domain provides a selective barrier that allows for absorption of nutrients, while it also forms a protective barrier against translocation of gut lumenal contents. The selective barrier function of the apical domain is modulated by microvilli and apical junctions. In intestinal disease, dysfunction of the apical domain alters its selective barrier function: malabsorption by microvilli is evident in celiac disease and microvillous inclusion disease, and disruption of the apical junction barrier contributes to the pathogenesis of Crohn's disease and infection by intestinal pathogens. The long-term objective of this research is to understand the process of apical domain assembly in intestinal disease. Thus far, there are no therapies that directly target apical domain assembly or disassembly. Filopodia are actin-based structures theorized to function in the assembly of microvilli and apical junctions. Myosin-X is a molecular motor that is required for filopodial formation, thus leading to the hypothesis that myosin-X functions in apical domain assembly. Preliminary lentivirus RNA interference studies strongly suggest myosin-X is necessary for microvillar formation, and preliminary observations may suggest a role for myosin-X in junctional protein localization. The specific aims of this proposal are two-fold: to determine the role of myosin-X and filopodia in the formation of microvilli; and to determine the role of myosin-X in apical junctional complex (AJC) assembly. Using Caco-2 cells as a model system, the localization and dynamics of myosin-X in apical domain structures will be determined by confocal microscopy and live-cell imaging. The hypothesis that myosin-X is necessary for microvillar formation will be tested in a stable myosin-X knockdown/rescue system. To test whether myosin-X is needed for AJC formation, AJC assembly will be induced by calcium switch in myosin-X knockdown cells. This study will determine the functional roles of myosin-X at the apical domain of intestinal cells and will reveal a novel molecular mechanism of apical domain formation. The proposed experiments will further our understanding of apical domain assembly, which is a necessary first step to address apical domain dysfunction in intestinal disease.